This is a proposal to investigate the role of stem cell regulation in cranial suture development and maintenance, and its pathophysiology in craniosynostosis. More broadly, this proposal focuses on how stem cells are controlled in space and time to promote the development and maintenance of vertebrate organs. Our recent results show that heterozygous loss of either of two related transcription factors, TWIST1 and TCF12, account for coronal suture defects in the majority of Saethre-Chotzen patients. Using sophisticated in vivo imaging and genetics in mice and zebrafish, we will test that Tcf12 modifies the function of Twist1 to maintain skeletal progenitors during both the specification and maintenance of sutures. A common role for Twist1 and Tcf12 in the developing and postnatal coronal suture would have the potential to explain both the initial synostosis and the high recurrence rate of postoperative synostosis in patients. A particular strength of our research plan is the complementary expertise of three accomplished investigators in craniofacial genetics. Rob Maxson has long-standing expertise in mouse models of synostosis, having contributed to the identification of TWIST1 and TCF12 as the two most affected genes in Saethre-Chotzen syndrome. Yang Chai recently identified a population of Gli1+ stem cells in the suture that are required for long-term suture patency and calvarial bone growth. Gage Crump has pioneered in vivo imaging techniques in zebrafish to examine the cellular basis of craniofacial defects. First, this team will test that Twist1 and Tcf12 function in the same tissues to repress the Ihh-driven differentiation of sutural progenitors into osteoblasts, as predicted if Tcf12 serves as a suture-specific dimerization partner for Twist1. Second, we will examine continuous requirements for Twist1 and Tcf12 in suture maintenance by conditionally deleting these genes in postnatal Gli1+ sutural stem cells. Third, we will use new knock-in tagged alleles of Twist1 and Tcf12 to identify the direct genomic targets of Twist1-Tcf12 dimers in postnatal sutural stem cells, as well as how Tcf12 modifies the ability of Twist1 to engage regulatory regions necessary for suture maintenance. Fourth, we will use powerful imaging techniques to reveal the in vivo spatial patterns of Twist1-Tcf12 dimers within sutures. Fifth, we will take advantage of the first zebrafish model of Saethre-Chotzen syndrome to directly visualize over time how changes in the pattern and timing of osteoblast differentiation result in later coronal suture defects. The results of these aims will test our model that Tcf12 functions as a suture-specific partner for Twist1, in part by guiding Twist1 to particular genomic regions necessary to inhibit premature osteoblast differentiation in suture mesenchyme. These new insights into the long-term requirements of synostosis genes in suture maintenance will have the potential to lead to new ways of preventing post-operative synostosis, thus reducing the number of risky operations currently performed on young children with Saethre-Chotzen syndrome.